The present invention relates to a method for preparing novel transcription factors which modify the procedure of transcription of gene information in a messenger RNA and their use.
The leading cause of death in developed countries other than Japan is ischemic heart disease. Further, in Japan cancer is the leading cause of death. In the United States of America, ischemia heart disease occupied 60 to 70% of the causes of death for a long time but as the result of the enlightenment movement on an extensive scale by the government and medical institutions, the death due to ischemia heart disease was reduced in an about 40% range. Even at present, however, ischemic heart disease is the first cause of death and occupies 70% of causes of death in the Scandinavian countries such as Sweden and Norway to run ahead of others. Further, in developing countries such as China, the top of the causes of death is ischemic heart disease, and regardless of industrialized countries and developing countries, the development of methods of the therapy and prophylaxis of ischemic heart disease as well as malignant tumor, type II diabetes, cerebrovascular diseases, obesity and the like is rightly an urgent need.
Ischemic heart disease of a representative life style-related disease frequently causes sudden death, and thus is the target of fear for people in highly industrialized countries. Three major risk factors which cause its onset are (1) a hereditary predisposition, (2) an environmental factor such as smoking, lipid metabolic disorder and diet and (3) aging. Of them, the hereditary disposition and the aging are very difficult to control. On the other hand, a large majority of highly industrialized countries continues making an effort to socially reduce environmental risk-factors. For example, smoking is regarded as a bad habit, and tobacco is banished from the environment, and suits against tobacco companies are frequently started.
As to the therapy/prophylaxis of ischemic heart disease, only the environmental factor is controllable. Then, for the purpose of removing risk-factors from the environment, a number of methods such as (1) diet therapy, (2) excercise therapy and (3) drugs are advocated. As to the diet, people in developed countries come to avoid intake of foods containing a large amount of cholesterol. Beef and eggs are victimized as the representatives of such foods and their consumption tends to be decreased over a long period of time. Excess intake of energy due to overeating induces obesity. It has been clarified that obesity alone cannot become a risk-factor but obesity combined with insulin resistance (type II diabetes), hyperlipidemia or hypertension becomes to be a risk-factor which is called as syndrome X (also known as silent death syndrome, deadly quartet or visceral obesity) (Reaven G. M, Diabetes 37:159-1607, 1988). In the United States of America, in order to avoid obesity, exercise therapy such as jogging and fitness which accelerate the consumption of energy is prevailing.
Of environmental risks which cause ischemic heart disease, the serum cholesterol concentration shows the highest correlation. The serum cholesterol concentration as a risk-factor is shown by several criteria. In other words, the criterion is whether or not the serum cholesterol or the cholesterol present in serum low density lipoprotein (LDL) is higher than the normal value or whether or not the serum high density lipoprotein (HDL) is lower than the normal value. It is known that in both cases, the onset risk of ischemic heart disease increases depending on the concentration. Accordingly, the drug therapy of reducing hypercholesterolemia with serum cholesterol lowering agents is also adopted as the standard therapy preventing ischemia heart disease. The serum cholesterol lowering agents have had their effect so that they had come to the most successful drug among therapeutic agents. Above all, an inhibitor of hydroxyl-methylglutaryl coenzyme A (HMG-CoA) reductase which reduces hydroxymethylglutaryl coenzyme A to mevalonyl coenzyme A is the most successful drug which is the top sales among the global drug market. The annual turnover of statin has reached 40 billion US Dollars or more.
On the other hand, the elucidation of the mechanism has advanced how angina pectoris and myocardial infarction are caused. Up to the 1980s, the medical professionals thought that cholesterol accumulated into the arterial intima formed atheroma plaques that constrict/block the intravascular cavity leading to ischemia of myocardium. However, since the 1970s, several clinical studies had been performed based on the hypothesis that it is a thrombus which constricts/blocks the coronary blood flow and prevention of the formation of the thrombus could prevent/treat ischemic heart disease. For example, such clinical trials used aspirin which is an anti-inflammatory agent and has an action of inhibiting plateletaggregation. Since aspirin induces a peptic ulcer during its chronic administration, it is very difficult to set dossier. In a long-term double blind clinical trials, aspirin was effective in some cases and was invalid in another trial, and thus aspirin has failed to obtain constant beneficial effect. After all, it seems likely that aspirin requires much higher doses to prevent arterial inflammation which frequently causes peptic ulcer and the smaller dose is not effective.
In the 1990s, instead of the concept that atheroma causes coronary ischemia, the hypothesis is presented that intra arterial blood vessel(s) induced by angiogenesi in atheroma rupture by inflammation and resulting blood clot occludes the blood stream. In other words, the hypothesis that inflammation of intra-arterial blood vessel(s) formed to nourish atheroma plaque(s) is causative of coronary heart disease (Ross R.: New England J. Med. 340:115-126, 1999) has been generally accepted. Therefore, the two major risk factors for coronary heart disease are hypercholesterolemia and chronic inflammation in atheroma plaque(s). It has been clarified from the epidemiological study that when the serum total cholesterol/HDL cholesterol level and the serum CRP level, the marker of chronic inflammation are both abnormally, the ratio of risk for myocardia infarction and cerebrovascular accidents are eight times higher than those being normal.
There is a therapeutic agent for improving the above described four disorders. However, as is clear from the fact that the co-administration of celibastatin-gemfibrozil induced rhabdomyolysis to threat lives, combined use of drugs may cause a danger of bringing about complicated interactions with each other. Thus, the number of drugs to be administered is preferably reduced. However, there is no drug which can treat the above described four disorders with single therapy.
There have been a number of therapies for the treatment of type II diabetes which does not requires daily insulin injections. It is known that type II diabetic patients frequently associate obesity at the onset, and its onset and obesity show a high correlation. Further, at the early stage of its onset, the obese patients show hyperinsulinemia and the type II diabetes is caused by the insufficiency in insulin action but not by that in its amount. Because the peripheral tissues in type II diabetes become resistant to the action of insulin. The first choice in treatment is diet therapy. Exercising enhances the peripheral sensitivity of insulin and is used together with the diet therapy.
If the cause of the onset and aggravation of type II diabetes are due to the insulin resistance in the peripheral tissue, the potentiation of the insulin sensitivity improves type II diabetes. In fact, one of the inventors has proved that 4-O-carboxymethylasco-chlorin (AS-6) of one of ascochlorin derivatives improves in the carbohydrate metabolism in hereditary obese diabetic mouse C57BL ksj (db/db), and the improvement is caused by the reduction in the insulin resistance of white adipose tissue (Hosokawa, Ando and Tamura; Diabetes, 34:267-274, 1985). Furthermore, the individual blood sugar levels of an AS-6 administered group and an AS-6 non-administered group and normal siblings and the carbohyrate metabolic capacity in the white adipose tissue are inversely correlated with a high correlation coefficient (r=−0.899), that is, the insulin resistance of the db/db mouse is caused by the disorder of energy metabolism of white adipose tissue and the improvement of energy metabolism of white adipose tissue by AS-6 reduces its insulin resistance. At present, it is clarified that when triglyceride is highly accumulated in the white adipose tissue, insulin resistance factors such as tumor necrosis factor-α and interleukin-1β which white adipocytes produce (Hotamisligil G. S., Sargaill N. S., Spiegelman B. M. et al: Science 259, 87-91, 1993) trigger the onset of type II diabetes. That is, it has been clarified that the white adipose tissue as well as the pancreatic Langerhans islet β cell is the target for the therapy of type II diabetes. Early in the mid-1980, however, the white adipose tissue was thought a mere store for triglyceridet and it was not anticipated that white adipose tissue plays a central role for a carbohydrate metabolism.
On the other hand, when attention is paid to cancer, it is clear that “surgical operation”, “anticancer agent” and “radiotherapy” called as the three major therapies are not always satisfactory under the present situation. Above all, anticancer agents which lead to complete cure are rare in spite of the long history of anticancer chemotherapy. The representative cancers which anticancer agents can lead to complete cure are acute myelocytic leukemia, malignant lymphoma, childhood viral tumor and the like. The beneficial effect of anticancer agents cannot be expected for prostatic cancer, breast cancer, large bowel cancer, gastric cancer, hepatoma, pancreas cancer, brain tumor and the like which account for a large majority of cancers. Furthermore, most anticancer agents are highly cytotoxic and the cells which propagate vividly are more sensitive to anticancer chemotherapy nonspecifically. Thus, anticancer agents have a side effect of inhibiting the multiplication of epithelial cells of the small intestine and reducing leukocytes to lower immune potency.
A greater problem for anticancer chemotherapy is that cancer cells readily acquire resistance to anticancer agents. The anticancer chemotherapies used in combination. However, cancer acquires resistance to any combination of anticancer agents in a short period of time and the anticancer effect is normally lost. Although the mechanism for the acquisition of resistance to anticancer agents is being studied, no means to clinically avoid the resistance has been found.
It is known that the ascochlorin and its derivatives have a time-dependent efficacy. The condition of exhibiting the drug efficacy is that the blood concentration continues over the threshold but not toxic concentrations for a definite period of time. For example, when AS-6 is orally administered to an animal, its blood concentration rapidly rises over the effective concentration approaching up to the toxic concentrations. Along with this defect, AS-6 is quickly excreted, the short duration of threshold concentration and the oxidation of aldehyde group to a carboxylic acid are weak points of this derivative. The carboxylic acid of AS-6 is less effective in efficacy as compared to AS-6. Further, there is a defect such that since the blood concentration of AS-6 quickly rises up to a concentration of exhibiting toxicity, the toxicity to the liver is easily exhibited.
4-O-Methylascochlorin (MAC) of another ascochlorin derivative developed prior to AS-6 has a solubility in water less than 0.7 μg/ml and is extremely sparingly soluble in water, and additionally very poor bioavailability due to the low water solubility has been a disadvantage in exhibiting the drug efficacy. Thus, the orally administered MAC mostly passes right through the gastrointestinal tract and is excreted in feces. Since the duration of the effective blood concentration is short and the peak of the blood concentration is low in animals, the defect of MAC is that the efficacy, for example, the lowering rates of the serum total cholesterol, blood sugar are low.
In other words, in order for ascochlorin and its derivatives to exhibit their efficacy, the prerequisites are less toxicity and higher drug-efficacy than hitherto ascochlorin derivatives; that is the derivatives continue the blood concentrations over threshold and below toxic levels for a definite period of time.
It is the most important to investigate and develop therapeutic agents for improving the syndrome X is the search for single compound efficacious for hypercholesterolemia, hypertension, hyperglycemia. Furthermore, it is also one of the problems to develop anticancer agents which do not reduce immune potency and do not allow cancer cells to acquire resistance. 4-O-Methylascocholrin and 4-O-carboxymethylascochorin for which the present inventors obtained a patent are the derivatives by modifying the phenolic hydroxyl group at the 4-position of the orcylaldehyde in ascochlorin. More than twenty years have passed since acquisition of the patents for these derivatives. Further, a number of the derivatives obtained by modifying the hydroxyl group at the 4-position of the orcylaldehyde in ascochlorin with an alky group or an allyl group have been synthesized, and thus it would be difficult to synthesize novel derivatives merely by substituting the hydroxyl group at the 4-position of the aromatic ring which could be patented.
The ascochlorin series compounds have a property of exhibiting time-dependent efficacy. That is, the blood concentration of an active substance has to be lower than the concentration of exhibiting toxicity and, at the same time, has to continue for a definite period of time above the threshold of exhibiting the drug efficacy. On the other hand, 4-O-alkylascochlorins obtained by alkylating the hydroxyl group at the 4-position of ascochlorin which have been studied heretofore are hardly soluble in water due to their high fat-solubility. Additionally, the rate of dissolution of the molecule in water from the crystal lattice is extremely slow, and when the 4-O-alkylascochlorins are orally administered to a small animal such as a rat and a mouse at fasting stage, they mostly pass right through the gastrointestinal tract and are excreted in feces. In addition to the low bioavailability, their absorption from the gastrointestinal tract also varies depending on the presence or absence of food intake (Agr. Bio. Chem., 46: 775-781, 1982) because bile secreted by food intake stimulates the rate of their solubility in water. Poor reproducibility in animal experiments has been an obstacle to practical applications of the 4-Oalkylascochlorins.
The rate of dissolution of the molecule in water from the crystal lattice can be expedited by introducing a polar group in the molecule. In fact, 4-O-carboxymethylascocholorin obtained by substituting the hydrogen at the 4-position of ascochlorin is soluble in water at a concentration of 6% or more at a pH of 7.2 to 7.7 in the small intestine, and thus is quickly absorbed on oral administration. As a result, there has been a defect of exceeding the blood concentration of exhibiting toxicity in a human and an animal.